HYPOTHESIS AND THEORY

published: 18 October 2016

doi: 10.3389/fpls.2016.01490

How Very-Long-Chain Fatty Acids

Could Signal Stressful Conditions in
Plants?
Antoine De Bigault Du Granrut 1 and Jean-Luc Cacas 1, 2*
1
UMR1318 Institut National de la Recherche Agronomique-AgroParisTech, Centre Institut National de la Recherche
Agronomique de Versailles-Grignon, Institut Jean-Pierre Bourgin, Versailles, France, 2 Département Sciences de la Vie et
Santé, AgroParisTech, UFR de Physiologie Végétale, Paris, France

Although encountered in minor amounts in plant cells, very-long-chain fatty acids exert
crucial functions in developmental processes. When their levels are perturbed by means
of genetic approaches, marked phenotypic consequences that range from severe growth
retardation to embryo lethality was indeed reported. More recently, a growing body
of findings has also accumulated that points to a potential role for these lipids as
signals in governing both biotic and abiotic stress outcomes. In the present work, we
discuss the latter theory and explore the ins and outs of very-long-chain fatty acid-based
signaling in response to stress, with an attempt to reconcile two supposedly antagonistic
parameters: the insoluble nature of fatty acids and their signaling function. To explain this
apparent dilemma, we provide new interpretations of pre-existing data based on the fact
Edited by:
Sylvain Jeandroz,
that sphingolipids are the main reservoir of very-long-chain fatty acids in leaves. Thus,
Agrosup Dijon, France three non-exclusive, molecular scenarii that involve these lipids as membrane-embedded
Reviewed by: and free entities are proposed.
Christina Kuehn,
Humboldt University of Berlin, Keywords: very-long-chain fatty acids, biotic and abiotic stress, signaling cascades, sphingolipids, membrane
Germany microdomains, plasma membrane, endoplasmic reticulum, secretory pathway
Susanne Hoffmann-Benning,
Michigan State University, USA

*Correspondence:
INTRODUCTION
Jean-Luc Cacas
jean-luc.cacas@agroparistech.fr; Both abiotic and biotic stresses, as well as developmental cues, have long been known to drastically
cacasjl@yahoo.fr modify lipid composition—including fatty acid (FA) content—at the organ level. For instance,
it is well-documented that phosphate starvation reorients lipid anabolism from phospholipid
Specialty section: toward galactolipid synthesis (Kobayashi et al., 2006), likely for maintaining plant cell homeostasis
This article was submitted to until the constraint is relieved. Likewise, temperature-induced stress provokes changes in plasma
Plant Physiology, membrane (PM) physico-chemical properties due to modification of sterol concentration and
a section of the journal FA double bond index (Los and Murata, 2004). Progressive loss of chloroplast galactolipids is
Frontiers in Plant Science
also a well-defined hallmark of foliar senescence processes (Jia and Li, 2015). Furthermore, plant
Received: 02 July 2016 resistance to pathogens can cause the consumption of chloroplast-originating polyunsaturated
Accepted: 20 September 2016 fatty acids for supplying an oxidative pathway that orchestrates host cell dismantling (Cacas
Published: 18 October 2016
et al., 2005). Obviously, all these events, whether or not associated with stress acclimation, are
Citation: relevant to profound structural alterations and mobilize huge amounts of lipids that can be
De Bigault Du Granrut A and
readily quantified by regular biochemical methods. By contrast, one can easily imagine that lipid-
Cacas J-L (2016) How
Very-Long-Chain Fatty Acids Could
contingent signaling events relies on more subtle changes. This is perfectly illustrated by the case of
Signal Stressful Conditions in Plants? phosphatidic acid, a conserved stress signaling molecule produced by either phospholipase D or the
Front. Plant Sci. 7:1490. coordinated action of phospholipase C and diacylglycerol kinase (Guo et al., 2011). Because of its
doi: 10.3389/fpls.2016.01490 low abundance, phosphatidic acid is commonly evidenced by in vivo isotopic labeling experiments

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De Bigault Du Granrut and Cacas VLCFA-Mediated Signaling of Stress

(Arisz et al., 2009; Cacas et al., 2016a). Another example that reductase (ECR). Each of these enzymes utilizes as substrate the
could be cited is that of the FA-derived hormonal signal jasmonic product of the previous one in cycles beginning by malonyl-
acid that requires highly sensitive liquid chromatography-based CoA condensation to long-chain acyl-CoA (Figure 1). Except
methods for efficient quantification (Glauser and Wolfender, for ECR, which is a single copy-encoded gene in Arabidopsis
2013; Cacas et al., 2016b). Additionally, to the best of our thaliana, a huge multigenic family composed of 21 members
knowledge, marked degradation of the respective lipid substrates codes for tissue-specific KCS enzymes (Joubès et al., 2008) that
alimenting the two latter signaling cascades were rarely correlated are thought to dictate the length of acyl-CoA chains produced
with signal generation. Hence, this hints the importance of by the complex (Fehling and Mukherjee, 1991; Millar and Kunst,
carefully considering, whenever possible, absolute concentrations 1997). Both KCR and HCD are encoded by 2 independent genes,
of metabolites involved when discriminating among signaling dubbed KCS1/KCS2 and PASTICCINO2/PTPLA, respectively
events and structural changes. What about very-long-chain fatty (Table 1). Such a complexity could suggest that multiple elongase
acids (VLCFA)? How are they synthesized? And, how their levels complexes, which differ by their relative composition, coexist
are affected in response to stress? in ER membranes. In other words, functionally spatialized-
domains with large metabolon units could orient the lipid class
into which very-long-acyl chains are incorporated. But, only
BIOSYNTHESIS OF VERY-LONG-CHAIN indirect evidence for this kind of ER sub-compartmentation
FATTY ACIDS IN PLANT CELLS were reported so far (Shockey et al., 2006). VLCFA are mainly
present in the impermeable cuticular wax layer deposited at the
In plants, lipid metabolism is highly compartmentalized and plant aerial organ surface, in triacylglycerides found in seed oil
this intricate organellar networks allows fine-tuned regulation and in sphingolipids, which act as structural elements in lipid
of the intracellular catabolic/anabolic balance for approximately bilayers forming endomembranes and PM (Bach and Faure,
several thousands of molecular lipid species. Biosynthesis 2010).
of FA-containing lipids—mostly phospholipids, galactolipids,
sphingolipids, triacylglycerides, and to a lesser extent, acylsteryl-
glycosides—relies on two interacting metabolic routes: the CHANGES IN VERY-LONG-CHAIN FATTY
“prokaryotic pathway” that resides in plastids and the “eukaryotic ACID LEVELS IN PLANT CELLS
pathway” that is localized to endoplasmic reticulum (ER).
Basically, production of FA-building units is initiated in plastids
UNDERGOING STRESS
by the fatty acid synthase (FAS) complex II that uses malonyl- With respect to modifications of VLCFA concentrations under
CoA and acetyl-CoA as co-substrates and NADPH as reductant. stressful conditions, sparse data have been obtained but clear
Each FAS-mediated cycle adds 2 carbons to acyl-CoA chains trends are currently emerging (Table 2). Overall, numerous
until molecules reaches a length of 16 or 18 carbons. Combined abiotic constraints (salt, cold, hypoxia, heavy metal exposure...)
thioesterase and acyl-CoA synthetase activities are then invoked were reported to increase VLCFA contents in distinct plant
in active export of aliphatic chains from stroma to cytoplasm, species. Induced Arabidopsis resistance to bacterial pathogens
where this pool of activated molecules is used by the ER for seems also associated with an augmentation of endogenous
further chain length extension (Li-Beisson et al., 2010). VLCFA levels (Raffaele et al., 2008). Not surprisingly, detailed
Very-long-chain fatty acids, formally defined as FA longer lipid analysis revealed that VLCFA, which are both components
than 18 carbons, are extended by an ER membrane-embedded and precursors of epicuticular wax, are affected by drought stress
protein complex of 4 enzymes, acting presumably on the and bacterial infection in proportions which are clearly relevant
cytosolic side (see Haslam and Kunst, 2013 for an updated to structural changes (Raffaele et al., 2008; Zhu and Xiong, 2013).
review). Fatty acid elongase activity results in successive action This experimental fact makes full sense as cuticle is involved in
of β-ketoacyl-CoA synthase (KCS), β-ketoacyl-CoA reductase limiting stomata-independent evaporation in shoots, suggesting
(KCR), β-hydroxyacyl-CoA dehydratase (HCD), and enoyl-CoA a reinforcement of this hydrophobic layer under water stress. In
the context of pathogen invasion, strengthening the apoplastic
Abbreviations: ACBP, Acyl-CoA Binding Protein; ACD5/11, ACCELERATED
barrier is also a well-known defense phenomenon (Garcion et al.,
CELL DEATH 5/11; BI1, BAX INHIBITOR 1; Cer, ceramide; Cer-P, ceramide
phosphate; CerS, ceramide synthase; CoA, coenzyme A; DIM, detergent-insoluble 2014), believed to prevent further micro-organism penetration
membranes; ECR, enoyl-CoA reductase; EIX2, ETHYLENE-INDUCING and spreading.
XYLANASE 2; ER, endoplasmic reticulum; ES, endomembrane system; FAH1, Pioneering works pointed out the transcriptional activation
fatty acid hydroxylase 1; FFAR/GPAR, FREE FATTY ACID RECEPTORS/G of genes coding for members of the Arabidopsis ER-localized
PROTEIN-COUPLED RECEPTORS; FA, fatty acid; FAH, fatty acid hydroxylase;
FAS, fatty acid synthase; GA, Golgi apparatus; GFP, green fluorescent protein;
elongase complex in response to stress. It has been demonstrated
GIPC, glycosyl-inositolphosphoryl-ceramide; GluCer, glucosylceramide; HCD, that multiple KCS-encoding genes were responsive to light
hydroxyacyl-CoA dehydratase; (h)VLCFA, (2-hydoxy-)very-long-chain fatty acid; conditions, dehydration, salt, cold, and osmotic stresses (Joubès
IPCS, inositolphosphoryl-ceramide synthase; KCR, β-ketoacyl-CoA reductase; et al., 2008). Mutants deficient for the transcription factor
KCS, β-ketoacyl-CoA synthase; LCB, long-chain base; LCB-P, long-chain base MYB30 were proven to be unable to accumulate WT levels
phosphate; LOH, LAG ONE HOMOLOG; MPK6, MITOGEN-ACTIVATED
PROTEIN KINASE 6; NADPH, Nicotinamide Adénine Dinucléotide
of VLCFA under hypoxia (Xie et al., 2015). In addition,
Phosphate; PM, plasma membrane; PR1, PATHOGENESIS-RELATED 1; SPT, microarray experiments showed that 3 out of the 21 KCS genes
serine-palmitoyl-CoA transferase; TGN, trans-Golgi network. (KCS1, KCS2, and KCS10), one HCD gene (PASTICCINO 2)

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De Bigault Du Granrut and Cacas VLCFA-Mediated Signaling of Stress

FIGURE 1 | Scheme representation of very-long-chain fatty acid elongation. This process takes place on cytosolic side of the ER membrane. It is alimented by
acetyl-CoA and acyl-CoA originating from the cytoplasm-located glycolysis and plastid-resident FA elongation pathway, respectively. Plastids provides 16 or 18
carbon-long acyl-CoA (i.e., palmitoyl- and stearoyl-CoA) to be elongated. The first committed step to VLCFA elongation cycle is catalyzed by the β-ketoacyl-CoA
synthase (KCS), which condenses malonyl-CoA (synthesized by carboxylation of acetyl-CoA moieties) with palmitoyl- or stearoyl-CoA. Resulting products are then
reduced into β-hydroxacyl-CoA (step 2) before losing a molecule of water (step 3); the latter reaction of which is mediated by the β-hydroxacyl-CoA dehydratase
(HCD). Upon dehydration, β-enoyl-CoA undergo reduction (step 4), forming acyl-CoA that harbor two additional carbons. These products can either be oriented
toward sphingolipid, wax and triacylglyceride synthesis depending on tissue specificity and cell requirement or reenter VLCFA elongation cycle until its length reaches
28 carbons in Arabidopsis or more in other plant species. Each cycle turn consumes one molecule of ATP and two of NADPH+H+ .

and the only ECR gene (CER10) were transcriptionally up- are several lines of evidence. Firstly, concentrations of VLCFA
regulated during incompatible interaction with bacteria, and mobilized in many instances described in the literature are all
the consecutive increase in VLCFA levels was confirmed by the more sufficient for signaling purposes (Table 2). Secondly,
biochemical approach. This transcriptional reprogramming was except for drought stress (Zhu and Xiong, 2013), no data can
further shown to be under the control of MYB30 (Raffaele et al., currently explain clearly the role of VLCFA in certain specific
2008). Although elongase regulation could account for cuticle abiotic contexts (like cold stress, mechanical injury and others)
structure readjustment, one cannot rule out the possibility that by the solely bias of cuticle. Thirdly, other lipids than wax
it could reflect an unusual context where VLCFA-contingent components, such as complex sphingolipids that are potential
changes hide signaling cascades. Arguing in favor of this idea reservoirs of signal molecules (Gronnier et al., 2016), exhibit

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De Bigault Du Granrut and Cacas VLCFA-Mediated Signaling of Stress

TABLE 1 | Nomenclature of the VLCFA elongase complex-encoding genes.

Gene names Other gene names Loci (AGI) Protein activity Protein length (aa) M.W. (kDa) pI

KCS1 - At1g01120 β-ketoacyl-CoA synthase (catalyzes the first 528 59.28 8.9
committed step to VLCFA synthesis)
KCS2 - At1g04220 528 59.53 9.6
KCS3 - At1g07720 478 54.33 9.5
KCS4 - At1g19440 516 57.84 9.1
KCS5 CER60 At1g25450 492 55.65 8.9
KCS6 CER6, CUT1, POP1 At1g68530 497 56.40 9.1
KCS7 - At1g71160 460 51.50 8.3
KCS8 - At2g15090 481 54.19 9.4
KCS9 - At2g16280 512 57.97 9.4
KCS10 FDH At2g26250 550 61.96 9.3
KCS11 - At2g26640 509 57.81 9.6
KCS12 - At2g28630 476 53.97 9.0
KCS13 HIC At2g46720 466 52.18 9.3
KCS14 - At3g10280 459 51.63 9.4
KCS15 - At3g52160 451 51.11 9.7
KCS16 - At4g34250 493 55.78 9.1
KCS17 - At4g34510 487 54.91 9.7
KCS18 FAE1 At4g34520 506 56.26 9.8
KCS19 - At5g04530 464 52.61 8.6
KCS20 - At5g43760 529 59.31 9.2
KCS21 - At5g49070 464 52.56 9.3

KCR1 - At1g67730 ketoacyl-CoA reductase 318 35.76 9.9

KCR2 - At1g24470 312 35.00 9.8

HCD PAS2 At5g10480 β-hydroxyacyl-CoA dehydratase 230 26.41 9.7

PTPLA - At5g59770 272 30.96 10

ECR CER10, GLH6 At3g55360 enoyl-CoA reductase 310 35.72 9.7

This table provides information on regular gene names, additional designations found in the literature and referenced loci based on the Arabidopsis Genome Initiative (AGI). Demonstrated
or potential enzymatic activities of the corresponding proteins are also indicated. Protein length is expressed as the number of amino acids (aa). M.W. and pI refers to molecular
weight and isoelectric point, respectively. Most data were retrieved from The Arabidopsis Information Resource (TAIR) website (https://www.arabidopsis.org/) and crossed with the
Arabidopsis book chapter dedicated to acyl-lipid metabolism (Li-Beisson et al., 2010). Abbreviations: CER6, 10 and 60, ECERIFERUM 6, 10 and 60; CUT1, CUTICULAR 1; FAE1,
FATTY ACID ELONGATION 1; FDH, FIDDLEHEAD; GLH6, GLASSY HAIR 6; HIC, HIGH CARBON DIOXIDE; PAS2, PASTICCINO 2; POP1, POLLEN-PISTIL INCOMPATIBILITY 1; PTPLA,
PROTEIN-TYROSINE PHOSPHATASE-LIKE.

significant changes in their VLCFA contents following stress and tissue homeostasis by acting as signaling molecules
(Table 2). Lastly, transgenic lines that displayed VLCFA over- through autocrine or paracrine cell non-autonomous modes.
accumulation correlated with enhanced pathogen-contingent cell Extracellular free FA concentrations can be finely perceived by
death phenotype (Raffaele et al., 2008). Given that cuticle-related plasma membrane-localized protein receptors that discriminate
processes are unlikely to control programmed cell death, it must among chain lengths. These are named FREE FATTY ACID
be envisaged that VLCFA exert their putative effects on cell fate RECEPTORS (FFAR)/G PROTEIN-COUPLED RECEPTORS
in an alternative manner. Thus, it seems reasonable to investigate (GPAR) (Ichimura et al., 2014). In plant genomes, no genes
the concept that VLCFA could participate to stress signaling coding for such orthologous receptors could be retrieved by
pathway. sequence comparison (unpublished data). Besides, even when a
number of long-chain acyl-CoA binding proteins (ACBP) were
reported to participate in plant stress tolerance (Xiao and Chye,
ARE FREE VERY-LONG-CHAIN FATTY 2011), it seems that they rather function as general regulators
ACIDS GENUINE SIGNALING of lipid metabolism than as cognate signaling partners of acyl
MOLECULES? chains in challenged cells. For instance, AtACBP2 and AtACBP4
were found to physically interact with an ethylene-responsive
In humans, lipid homeostasis is tightly controlled, and its transcription factor (Li and Chye, 2004; Li et al., 2008), possibly
long-term perturbation can have severe deleterious effects controlling by this means lipid-related gene expression. Another
on health. Free FA contribute to the regulation of organ issue for VLCFA to be considered as genuine signals relies on

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De Bigault Du Granrut and Cacas VLCFA-Mediated Signaling of Stress

TABLE 2 | Changes in VLCFA levels under stressful conditions.

Plant models Stresses FA phenotypes Affected lipid classes* Analyzed organs References

Nigella sativa L. Mild Zn2+ exposure Increase in 20:0, 22:0 and n.d. Seeds Marichali et al., 2016
24:0
Increase in 20:0, n.d. Leaves
22:0 and 24:0 Decrease in
20:1
Increase in 20:1, n.d. Stems
22:0 and 24:0 Decrease in
20:0
Increase in 20:0, n.d. Roots
22:0 and 24:0 Decrease in
20:1

Noccaea caerulescens, Cd2+ exposure Decrease 26:0, n.d. - Zemanová et al., 2015
ecotype Mezica 28:0 and 30:0
Increase in 20:2 and 20:3

Tetraselmis sp. M8 Salt Increase in 20:4 and 20:5 n.d. - Adarme-Vega et al., 2014

Artemisia annua Long-term salinity Decrease in 22:0 and 24:0 n.d. Leaves Qureshi et al., 2013
Increase in 22:1

Taxus chinensis cv. Shear stress Increase in 20:0, 20:1, n.d. Suspension cell culture Han et al., 2009
mairei 22:0, 24:0 and 25:0 from stem

Rice (Oriza sativa) Drought Increase in 26:0 and 28:0 Cuticular wax Leaves Zhu and Xiong, 2013

Arabidopsis thaliana Hypoxia Increase in 22:0, GIPC and GluCer Leaves Xie et al., 2015
24:0 and 24:1

Arabidopsis thaliana Oxidative stress Increase in total hVLCFA n.d. Leaves Nagano et al., 2012

Arabidopsis thaliana Cold n.d. Increase in GIPC Shoots Nagano et al., 2014

Arabidopsis thaliana Pst DC3000::AvrRpm1 Total VLCFA n.d. Leaves Raffaele et al., 2008

This table provides an overview of selected publications dealing with abiotic stress. Of note, to the best of our knowledge, the only published work reporting on biotic stress and VLCFA
content is that of Raffaele et al. (2008). Nomenclature for fatty acids (FA) is as follows: the first number indicates the carbon chain length and the second the number of unsaturation
(example: 24:1 is 24 carbon-long FA which possesses one double bond). n.d., not determined, * refers to the lipid classes whose VLCFA content is altered. Pst refers to Pseudomonas
syringae pv. tomato.

their amphipathic nature that renders them strong membrane more complex glycosylated sphingolipids, known as glycosyl-
destabilizers and not prone to cross lipid bilayers. This certainly inositolphosphoryl-ceramides (GIPC) (Figure 2A). Apart from
prevents unmodified VLCFA from functioning as soluble signals GIPC, the synthesis of which is initiated in the ER and completed
at both the intra—and extra-cellular levels. Therefore, alternative in the Golgi apparatus (GA), the three other classes are produced
hypotheses must be imagined for explaining how these lipids in the ER (Figure 2B). Neo-synthesis of LCB results from the
could regulate plant stress responses. What could be the condensation of serine and palmitoyl-CoA moieties catalyzed
molecular mechanisms invoked? Based on the literature, three by a protein complex, so-called the serine-palmitoyl-CoA
potentially interconnected scenarii taking into account the transferase (SPT). Subsequent reduction of the SPT product
observed relatively high amounts of VLCFA mobilized during results in the synthesis of sphinganine, the precursor of the
stress response are described hereafter. eight other LCB found in plants. Ceramide synthases (CerS),
encoded by a multigenic family named after the yeast protein
LAG ONE HOMOLOG (LOH), are responsible for the formation
“THE INDIRECT SPHINGOLIPID of the amide bond that links (V)LCFA to LCB, leading to
SIGNALING HYPOTHESES”–HOW TO Cer formation. Ceramides can then be used as backbone for
RECONCILE VERY-LONG-CHAIN FATTY the production of GluCer and GIPC by addition of a glucose
ACIDS WITH STRESS SIGNALING? molecule or an inositolphosphoryl group followed by one or
several glycosylation steps, respectively (Markham et al., 2013).
Plant sphingolipids encompass four major classes: long-chain Noteworthy, it can be inferred, on the basis of their biosynthetic
bases (LCB), ceramides (Cer), glucosylceramides (GluCer), and pathway (Li-Beisson et al., 2010), and additional data (Pata

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De Bigault Du Granrut and Cacas VLCFA-Mediated Signaling of Stress

FIGURE 2 | Plant sphingolipid synthesis. (A) The four classes of plant sphingolipids (from Cacas et al., 2012a). The left panel displays the nine molecular species
of long-chain bases found in plants, from top to bottom: sphinganine/dihydrosphingosine, d18:0; sphingosine/sphing-4(trans)-enine, d18:114(E) ;
sphing-8(trans)-enine, d18:118(E) ; sphing-8(cis)-enine, d18:118(Z) ; sphinga-4,8(trans, trans)-dienine, d18:114,8(E,E) ; sphinga-4,8(trans, cis)-dienine, d18:114,8(E,Z) ;
(Continued)

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De Bigault Du Granrut and Cacas VLCFA-Mediated Signaling of Stress

FIGURE 2 | Continued
phytosphingosine/4-hydroxysphinganine, t18:0; 4-hydroxysphing-8(trans)-enine, t18:118(E) ; 4-hydroxysphing-8(cis)-enine, t18:118(Z) . On the right panel (from top to
bottom) are showed a ceramide (sphing-4(trans)-enine-N-octadecanoic acid), a glucosylceramide (Glucosyl-O-β-ceramide
(sphing-4(trans)-enine-N-octadec-9(cis)-enoic acid)) and a glycosyl-inositolphosphoryl-ceramide (N-acetylglucosamine-glucuronic acid-inositolphosphoryl-ceramide
(4-hydroxysphing-8(cis)-enine-N-tetracosanoic acid)). (B) In situ simplified view of the plant sphingolipid biosynthesis pathway. Except for the serine and palmitoyl-CoA
precursors, sphingolipid metabolites appear in colored rectangles: green for long-chain bases, orange for ceramides and red for final products like glucosylceramides
(GluCer) and glycosyl-inositolphosphoryl-ceramides (GIPC). Nomenclature for ceramide is as follows: for instance, d18:0-16:0 indicates that the long-chain base
corresponds to sphinganine and the fatty acid is a palmitoyl moiety, respectively. Enzymes are written in red. Abbreviations: CerS, ceramide synthase; DAG,
diacylglycerol; FAHase , fatty acid hydroxylase; GCS, glucosylceramide synthase; GTase , glycosyl-transferase; IPCS, inositolphosphoryl-ceramide synthase; IPUT1,
INOSITOLPHOSPHORYL-CERAMIDE GLUCURONOSYL-TRANSFERASE 1; LCB, long-chain base; LCB DESase , LCB desaturase; LCB OHase , LCB hydroxylase;
LCFA, long-chain fatty acid; LOH, LAG ONE HOMOLOG; PI, phosphatidylinositol; SPT/FBR11, serine palmitoyl-CoA transferase/FUMONISIN-RESITANT 11; SLD1,2,
SPHINGOLIPID LCB 18 DESAUTRASE 1,2; UDP-Glc, uridine diphosphate-glucose; VLCFA, very-long-chain fatty acid.

et al., 2010; Cacas et al., 2016c), that sphingolipids contain most for those that overexpressed LOH2. Additional phenotypical
VLCFA produced in leaves. traits of the latter overexpressor plants were reminiscent
of lesion-mimic mutants that exhibit enhanced disease
Scenario 1: Interplay between resistance and develop hypersensitive cell death symptoms
Very-Long-Chain Fatty Acids and the under restrictive environment, despite the absence of pathogens.
These traits included increased salicylic acid concentrations and
Ceramide and LCB Signals PATHOGENESIS-RELATED 1 (PR1) gene expression, localized
Schematically, GIPC represent two third of total sphingolipids
programmed cell death and severe dwarfism (Luttgeharm
within photosynthetic plant cells whereas Glucer accounts for
et al., 2015). Consistently, loh1 null mutants displayed discrete
the other third (Markham et al., 2006). This is coherent with
spontaneous foliar lesions correlated with strong constitutive
their role as structural membrane elements. By contrast, free
PR1 expression and significant elevation in 16:0-containing Cer
Cer and LCB, as intermediate metabolites, are weakly present in
and GluCer contents (Ternes et al., 2011). Although FA-mediated
leaf organs (Markham et al., 2006). Defining genuine molecular
structural effects cannot totally be ruled out for explaining the
signals as locally- and timely-produced molecules that act at
aforementioned phenotypes (see below), these data may also
infinitesimal concentrations, it might not be surprising that the
pinpoint the importance of the aliphatic chain length present in
signaling function of both LCB and ceramides under stressful
Cer destined to signaling purposes, suggesting the occurrence
conditions could be conserved across kingdoms. Even though
of a supplementary regulatory level to that driven by the solely
exact molecular substratum for sphingolipid control of cell fate
balance of phosphorylated/non-phosphorylated metabolites.
is far from being deciphered, it is assumed that, in plants,
In line with this postulate, it is thus tempting to speculate
like in animals, accumulation of free Cer or LCB would kill
that stress-induced rise in VLCFA concentrations might alter
cells whereas that of their phosphorylated counterparts would
intracellular Cer pool composition and, subsequently, impact
have survival-promoting effects in response to stress. In plants,
related signaling routes. Moving further this way, it is also
when chemically-perturbed or genetically-disrupted, most steps
plausible that, following stress exposure, quantitative and/or
of the sphingolipid biosynthesis pathway can lead to conditional
qualitative modifications of VLCFA pool indirectly influence
cell death phenotypes or spontaneous pathogen resistance-
LCB synthesis due to metabolic reorientation, as suggested by
mimicking hypersensitive-like foliar lesions. These observations
the analysis of LCB hydroxylase mutants (Chen et al., 2008). The
can be tentatively explained by a disequilibrium of the tightly-
existence for two Arabidopsis sphingosine kinases with sharply
regulated intracellular balance between unfettered LCB and LCB-
different substrate specificity also reinforces the idea that both
phosphate (LCB-P). Compelling evidence for this notion was
VLCFA and LCB chains could matter when it comes to signaling
provided by exogenous LCB application, the use of the mycotoxin
stress (Guo et al., 2011). Since the Arabidopsis MITOGEN-
fumonisin B1 that inhibits CerS and mutation (fumonisin-
ACTIVATED PROTEIN KINASE 6 (MPK6) has been recently
resistant 11, fbr11) in a subunit of the LCB-forming enzyme
described as a downstream effector of sphingolipid-induced
SPT (Alden et al., 2011; Berkey et al., 2012). Another crucial
cell death (Saucedo-García et al., 2011), it could represent a
regulatory node may also rely on the Cer/Cer-phosphate (Cer-
privileged target for investigating the interplay between VLCFA
P) ratio, as substantiated by genetic data regarding the ceramide
and LCB/Cer-contingent signaling under stressful conditions.
kinase ACCELERATED CELL DEATH 5 (Liang et al., 2003), the
Cer-P transferase ACCELERATED CELL DEATH 11 (Simanshu
et al., 2014) and inositolphosphoryl-ceramide synthase (IPCS) Scenario 2: The Membrane Trafficking Link
(Wang et al., 2008). The plant endomembrane system (ES) is a complex, dynamic
Recent work addressing Arabidopsis CerS specificity toward and intricate membrane-composed web that encompasses the
FA chain length in vitro established that LOH1/3 preferentially ER, Golgi apparatus (GA), trans-Golgi network (TGN), the
use VLCFA as substrates whereas LOH2 rather forms sixteen endocytic, vacuolar, and autophagic compartments, the plasma
carbon-long fatty acid (16:0)-containing Cer (Luttgeharm membrane (PM) and all vesicles that shuttle in between these
et al., 2016). While LOH1/3 overexpressing lines showed organelles. It provides infrastructure for the secretory pathway
only little changes in their sphingolipid profiles, a strong which is dedicated to both protein and lipid sorting (Cacas, 2010).
enrichment in Cer molecular species with 16:0 FA was recorded Apart from its crucial role in maintaining cellular homeostasis,

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De Bigault Du Granrut and Cacas VLCFA-Mediated Signaling of Stress

the ES has also recently emerged as an essential component of sphingolipid synthesis was manipulated (Markham et al., 2011).
plant tolerance to stress (for review, see Cacas, 2015) and, this Together with those of Melser’s work, our results provide
may be linked to VLCFA-containing sphingolipids, like GluCer unequivocal evidence for the requirement of VLCFA-containing
and GIPC. sphingolipids for protein transport, even though the respective
Early indirect insights into a potential relationships between contribution of GluCer and GIPC in this process could not
GluCer and protein trafficking came from clinical studies focused have been ascertained. Back to signaling topic, it is possible that
on molecular mechanisms underpinning Gaucher disease. A VLCFA anabolism adjustment participate in accommodating cell
bench of mutations that totally or partially invalidate the secretory activity to challenging environmental conditions. In
two glucocerebrosidases-encoding genes—involved in lysosomal fact, several published examples already indicate that effective
degradation of GluCer—was found to cause ER to dysfunction, intracellular membrane trafficking is necessary for transducing
leading to enzyme sorting impairment (Yu et al., 2007). specific protein-based signals during plant immunity (for
In plants, regulation of GluCer homeostasis is as essential reviews, see Berkey et al., 2012; Teh and Hofius, 2014). Among
as in animal models. In Arabidopsis, only one gene codes them can be cited the immune receptor EIX2 (ETHYLENE-
for the ER-localized glucosylceramide synthase, or GCS. Null INDUCING XYLANASE 2) from tomato plants, the endosomal
gcs mutants fail to develop beyond seedling stage and are internalization of which is necessary for mounting proper defense
defective for organogenesis. In addition, gcs (−/−) cells display response (Sharfman et al., 2011). Another striking example is
altered GA morphology indicative of a probable perturbed cell that of the relocalization of the RPW8 resistance protein to
secretion activity (Msanne et al., 2015). Accordingly, ground- extrahaustorial membranes at the host-pathogen interface in
breaking work carried out by the team of Dr. Moreau (CNRS, response to specific fungi and oomycetes (Wang et al., 2009).
Bordeaux, France) pointed out that chemical blunting of GCS Moreover, numerous protein regulators that control cell death
activity resulted in (i) GA disaggregation into vesicles, (ii) outcome in response to abiotic and biotic stress are distributed
reduced externalization of an apoplastic fluorescent protein along the ES (Cacas, 2015). This sustains the idea that VLCFA
ectopically expressed (N-SecYFP), and (iii) both mislocalization could define a late secretory pathway dedicated to some stress
and secretion diminishment of the PM-located H+ -ATPase signaling components. One of the main challenges in next future
PMA4 (Melser et al., 2010). Transient overexpression of the will be to understand the role of one such path under abiotic
two latter proteins (N-SecYFP and PMA4) in a WT genetic constraints. Distinguishing how VLCFA could, respectively,
background was further reported to augment sterol and GluCer influence antimicrobial protein burden to be excreted and
contents whereas that of soluble proteins and membrane proteins transport of specific regulatory proteins following pathogen
which do not traffic beyond GA was unable to do so (Melser infection will probably represent a difficult task too.
et al., 2010). Combined, these findings put forward the case for
GluCer, along with sterols, as potent protein sorting mediators
in the late secretory pathway. Two main explanations can be Scenario 3: The Membrane Microdomain
envisaged in this context. On the one hand, one can assume that Hypothesis
GluCer and sterols exerts their function through a chaperone-like Membrane microdomains can be defined as islands composed
activity, stabilizing native structure of specific integral membrane of lipids and proteins that laterally segregate from the rest
cargo proteins that they escort from GA to PM. On the other of the PM. They are highly enriched in sterols, sphingolipids
hand, it has been proposed that glycosphingolipids could impose and signaling proteins (Boutté and Grebe, 2009). Their size
positive curvature to membranes, thereby facilitating vesicle was described to fit nanometer to micrometer scales. Originally
fusion (Barth et al., 2010; Molino et al., 2014). In this regard, the named raft, microdomains were identified in mammalian
few data that are currently available in the literature do not allow systems where they notably act as platforms responsible for
discriminating among these two hypotheses yet. the launching of apoptotic and inflammation signaling cascades
Joined study between our team (Faure’s lab, INRA, Versailles, (Malorni et al., 2007; George and Wu, 2012). First experimental
France) and that of Markham (Danforth Plant Science Center, evidence for their occurrence in plants was provided by
Saint Louis, Missouri, USA) documented the impact of loh biochemical approaches based on the purification of detergent-
mutations on Arabidopsis root architectural modifications in insoluble membranes (DIM) through floatation on step sucrose
relation with sphingolipid profile and secretion of PM-resident gradient (Mongrand et al., 2004). Since then, accumulation of
proteins (Markham et al., 2011). As corroborated latter on pharmacological, proteomic, microscopy, and genetic data ended
by Luttgeharm et al. (2016), it was demonstrated that double the controversy on plant microdomain existence. Thus, it is
loh1/loh3 mutants overaccumulated 16:0-containing complex now broadly accepted that DIM do not represent functional
glycosphingolipids at the expense of VLCFA-containing ones, equivalents of microdomains, but rather constitutes one way of
reflecting the substrate specificity of the remaining CerS activity assessing their chemical composition (Cacas et al., 2012a).
borne by LOH2. Remarkably, this marked trend was correlated Previously, a strong enrichment in tri-hydroxylated LCB
with PM-targeting default for two auxin carrier proteins; the in PM fractions purified from two plant species was reported
latter of which being characterized by a loss of cell polarity, (Borner et al., 2005; Lefebvre et al., 2007). Given that this
a strong inhibition of hormonal transport and the absence of class of LCB is mainly encountered in GIPC (for review, see
lateral root initiation at macroscopic level. Again, experimental Pata et al., 2010), this led the plant lipid community to the
data argued in favor of a post-Golgi trafficking defect when reasonable hypothesis that GIPC reside, for the most part,

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De Bigault Du Granrut and Cacas VLCFA-Mediated Signaling of Stress

in PM. Having established methods for purifying GIPC and pool, and (ii) VLCFA moieties engaged in these lipids were
characterizing their composition (Buré et al., 2011; Cacas et al., predominantly hydroxylated on carbon position 2 (noted
2012b), we tested for this assumption using tobacco plants hVLCFA). Exploiting this unique opportunity for probing GIPC
and cell cultures. Not astonishingly, we found that (i) tobacco repartition within the ES uncovered a marked hVLCFA gradient
GIPC contain the large majority of the intracellular VLCFA along the secretory pathway that reaches an optimum in DIM

FIGURE 3 | Molecular model explaining how very-long-chain fatty acids could participate in stress signaling response in plant cells. In mammalian
systems, extrinsic cues can be perceived at the plasma membrane by means of microdomains. A comparable hypothesis can be emitted for plant models. It is
possible that “sphingolipase D” (SPLase) like the one identified by Tanaka et al. (2013) is recruited to microdomains following stress exposure, and releases ceramide
(Cer) molecules from complex glycosphingolipids in situ. Free Cer could either directly act as signals or be processed into Cer-P by the kinase ACCELERATED CELL
DEATH 5 (ACD5). The ceramide-1-phosphate tranferase ACCELERATED CELL DEATH 11 (ACD11) may participate to this signaling cascade as well, though its exact
mode of action remains to be clarified. In addition, Cer could also be hydrolyzed by ceramidase (CDase) into LCB that can, in turn, be phosphorylated; one such
activity have indeed been documented in plants (Pata et al., 2010). Although little is still known about molecular actors that relay LCB/Cer signals (orange part of the
figure), the work of Saucedo-García et al. (2011) identified MITOGEN-ACTIVATED PROTEIN KINASE 6 (MPK6) as a good candidate for exerting this function. The
transcription factor MYB30 represents a potential downstream target of sphingolipid-induced phosphorylation events, since it was found to up-regulate acyl-CoA
elongase genes in response to environmental cues. Resulting very-long-chain fatty acid (VLCFA) production could then be utilized for strengthening cuticle, especially
epicuticular wax. Alternatively, VLCFA could be incorporated into sphingolipids. Modifications of sphingolipid composition and/or level can impact protein sorting at
the TGN and, therefore, probably modulate targeting of specific stress responsive signaling proteins to PM. From this postulate, it seems coherent to envisage that
modifications of the secreted lipids and proteins influence PM lateral segregation. Expected consequences of this segregation phenomenon could be changes in
microdomain content that could feature extracellular signaling process(es) and/or negative feedback regulation. Of note, elements in the picture that represent
regulatory nodes involving VLCFA (Hypotheses 1-3) are delineated by red continue or discontinued lines. Red discontinued arrows indicate steps which has not been
experimentally demonstrated. For additional abbreviations, refer to the legend of Figure 2.

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De Bigault Du Granrut and Cacas VLCFA-Mediated Signaling of Stress

fractions. Further investigations brought to light that GIPC described to promote rapid relocalization of sphingolipid-
amount to approximately 60 mole % of total DIM lipids; the modifying enzymes to microdomains, freeing Cer or LCB
polyglycosylated forms being only present in the external hemi- moieties and, per se, generating primary signals relayed by
layer and clustering in 35 nm-sized microdomains. Combined downstream effectors. Actually, one can expect that both
biophysical and modeling strategies also showed that hVLCFA situations could cohabit in the same challenged plant cell
could strongly interact with sterols and interdigitate between the with different timing. In this case, raft sphingolipids could
two membrane leaflets, which likely explains the synergistic effect feature a reservoir of signals to be mobilized early following
of GIPC and sterol in structuring membrane in vitro (Cacas et al., stress application, as sustained by the recent discovery of
2016c). Hence, in addition to their postulated role in protein sphingolipase D activities in plants (Tanaka et al., 2013). Once
sorting at the TGN, glycosphingolipids may also be involved in initiated, such signaling cascades would contribute to activate
stress response with respect to their lateral segregation within hVLCFA neo-synthesis, ultimately fine-tuning microdomain
PM. composition. The latter phenomena could either benefit to
Nagano et al. (2009, 2012) shed light on possible links between intercellular communication or simply operate as a negative
hVLCFA and stress acclimation. Working on the conserved feedback that abrogate the production of signaling molecules by
family of ER-resident cell death regulators, known as BAX microdomains. Testing for this seductive concept will require
INHIBITORS (BI), the authors showed that At-BI1 interacts a careful in situ dosage of hVLCFA-containing sphingolipids
with the electron donor, cytochrome b5, the latter of which over time. With the recent advances in mass spectrometry-based
can in turn associate with FATTY ACID HYDROXYLASE1 chemical imaging, this deadlock should be broken in a close
(FAH1) catalyzing the hydroxylation of VLCFA. Overexpression future.
of At-BI1 was also correlated with higher hVLCFA contents
and decreased cell death under stressful conditions. Conversely,
knock-down FAH1 plants displayed decreased hVLCFA amounts
TENTATIVE MODEL–PUZZLING OUT
and enhanced sensitivity to hydrogen peroxide, suggesting that VERY-LONG-CHAIN FATTY
At-BI1 protects cells by activating FAH1. Now, given that ACID-CONTINGENT SIGNALING
hCer exhibit pro-survival properties in animal cells (Young PATHWAYS
et al., 2013), one can hypothesize that the hCer/Cer balance
under the control of BI1/FAH1 dictates the progression rate of Although the involvement of VLCFA in stress response
hypersensitive foliar lesions in response to pathogen attack. This is not contestable, interpretation of this experimental fact
attractive theory cannot, however, justify by itself MYB30-driven may remain delicate in light of the currently available
transcriptional induction of the elongase complex genes (Raffaele data. Pleiotropic consequences of VLCFA level alterations
et al., 2008) and the resulting massive increase in VLCFA also render this task complicated. Based on the observation
concentrations reported in this context. Alternatively, one can that VLCFA are highly enriched in sphingolipids, we have,
envisage that modulating hVLCFA synthesis could affect GIPC however, suggested, and explored three non-exclusive, molecular
composition and/or concentration and, consequently, impact raft scenarii to tentatively explain how insoluble molecules —like
signaling events. In vivo and in vitro experiments have proven VLCFA—could participate in stress signaling response. These
that lateral segregation of membrane proteins is dependent hypotheses, which are experimentally testable, are summarized
on that of lipids, and vice versa (for review, see Volmer and in a model presented in the Figure 3. By analogy with
Ron, 2015). In animal systems, recruitment or disassembly of animals systems, it is envisaged that stress perception could
signaling actors can be achieved through respective coalescence trigger recruitment of yet-to-be cloned “sphingolipases” to
or dissociation of microdomains, thereby provoking initiation microdomains. Enzymatically-released Cer(-P) skeletons could
or termination of signaling cascades at the PM (Malorni et al., then either directly serve as signals or be further processed,
2007; George and Wu, 2012). A comparable situation has already activating a potent downstream effector, the kinase MPK6.
been proposed to take place in BI1-overexpressing transgenics One possible target of this phosphorylation cascade could
(Ishikawa et al., 2015) and during plant innate immunity be the transcription factor MYB30, which is known to up-
(Keinath et al., 2010). In addition, both biotic and abiotic regulate expression of the elongase complex-encoded genes upon
stresses are known to provoke changes in protein content of pathogen attack and hypoxia. As also supported by several
microdomains (Minami et al., 2009; Stanislas et al., 2009). studies, changes in the composition and/or level of VLCFA-
Findings that elongase complex-encoding genes are under containing lipids impact protein sorting at the TGN. This
transcriptional control upon environmental cues (Joubès et al., could represent a potential regulatory mechanism whereby
2008; Raffaele et al., 2008; Xie et al., 2015) implies that targeting of specific signaling proteins to PM could be spatio-
modulation of hVLCFA steady-state levels is implicated in a temporally modulated depending on stressed cell requirements.
secondary signaling wave, possibly regulating microdomain- Adding a supplemental layer of regulation, these changes
coordinated events. Indeed, for transcriptional reprogramming in sphingolipids certainly alter microdomain content, and
to occur, stress perception must be completed and signal consequently, should also influence PM-coordinated signaling
transduction engaged. This is quite distinct from, but not events. Beyond the response plasticity conferred to plant
incompatible with the regular picture documented in the cells by a dual lipid/protein-based rheostat, this model raises
mammalian literature where external constraints are generally the interesting question as to how this molecular scheme

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De Bigault Du Granrut and Cacas VLCFA-Mediated Signaling of Stress

contributes to stress acclimation. Is this linked to intercellular ACKNOWLEDGMENTS

communication, negative feedback control of microdomain-
dependent signaling or both? We would like to warmly thank Dr. Palauqui JC (INRA,
Versailles) for critical reading and enthusiastic discussions.
We also would like to thank the editors for inviting us
AUTHOR CONTRIBUTIONS contributing to this special issue. We declare no conflict of
interest with any works cited in this article. We apologize
AD provided Table 2, JC defined hypotheses, wrote the in advance for not citing colleagues’ research due to space
manuscript, and drew figures. limitation.

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